Few stroke laboratories study female animals or use cell models of ischemic brain injury that are sex-specific. In part, this is due to the historical assumption that cellular/molecular injury and repair mechanisms are the same in males vs. females. The persistent lack of pre-clinical animal data in both sexes poses a severe evidence gap for clinical trialists who will test new therapies in women and men. In this application, we test the overarching hypothesis that the evolution of post-ischemic inflammatory cycling between the brain and peripheral immune system is strongly influenced by biological sex, including sexually dimorphic immune cell subsets and key inflammatory mechanisms that affect brain-spleen-brain cycling of inflammatory cells after focal cerebral ischemia. Aim 1 will test the hypotheses that evolving cerebral ischemic injury elicits splenic damage in tandem with brain microvascular and parenchymal inflammation that is more profound and is mediated by different immunocyte populations (monocytes vs. T lymphocytes) in males vs. females. Aim 2 determines if T lymphocyte-mediated injury in post-ischemic brain is greater in females due to lower levels of peroxisome proliferator activated receptor alpha (PPAR) in T lymphocytes. These hypotheses predict that (a) while splenectomy benefits the injured brain of both sexes by eliminating immunocytes nurtured within the spleen, adoptive transfer of female vs. male T lymphocytes sensitized to focal cerebral ischemia into splenectomized same sex recipients selectively increases female cerebral damage more so than males following MCAO and that (b) female vulnerability to T lymphocyte-mediated injury is due in part to a lack of protective PPAR signaling mechanisms. Aim 3 will evaluate whether monocyte trafficking from spleen to post- ischemic brain is sex-specific. The hypotheses are that (a) males exhibit an early and more robust recruitment of CD45highCD11b+ macrophages and CD11c+ dendritic cells into post-ischemic brain relative to females; (b) this recruitment is due in part to higher matrix metalloproteinase (MMP)-9 expression in male monocytes, thus facilitating their transmigration; (c) male mice deficient in CD11b+ (macrophage knockout) or in CD11c+ myeloid cells (dendritic cell knockout) will more greatly benefit by loss of these cells than will females following focal cerebral ischemia. Findings from this application could therefore lead to the development of new therapies for stroke such as PPAR agonists for females and MMP-9 antagonists for males. PUBLIC HEALTH RELEVANCE: Ischemic stroke is a leading cause of death and disability in the United States, yet relatively little is known about the contribution to and effects on the immune system during stroke. The application focuses on early and late events in the peripheral immune system during stroke and important changes in the spleen that occur simultaneously with the evolving brain injury. We have preliminary evidence that this process is not identical in both males and females and are studying the relevant sex differences.